Novel method of inducing antigen-specific t cells

ABSTRACT

The present invention provides a novel method for inducing antigen-specific T cells. A method for inducing antigen-specific T cells in a patient comprising administering to said patient in need thereof composition (a) which comprises a therapeutically effective amount of an antigen protein or an antigen peptide as an active ingredient, and composition (b) which comprises a therapeutically effective amount of a cell wall skeleton integrant of the BCG strain of  Mycobacterium bovis  as an active ingredient, wherein composition (b) is administered in advance and then composition (a) is administered, and related pharmaceutical compositions are provided.

FILED OF THE INVENTION

The present invention relates to novel methods of inducingantigen-specific T cells. In particular, the present invention relatesto methods of inducing antigen-specific T cells, which administering acomposition comprising a cell wall skeleton integrant of the BCG strainof Mycobacterium bovis (hereinafter, as referred to as BCG-CWS) inadvance and then administering a composition comprising an antigenprotein or an antigen peptide. In addition, the present inventionrelates to compositions for treating and/or preventing cancers that arecharacterized in that they comprise a cancer antigen protein, WT1, or acancer antigen peptide derived from said WT1 protein in combination witha BCG-CWS.

BACKGROUND ART

Cellular immunity mediated by, among others, cytotoxic T cells (alsosometimes referred to as killer T cells or CTLs) or helper T cells,which are antigen-specific T cells, plays a central role in eliminationof cancer cells or virus-infected cells from a living body. Anantigen-specific T cell recognizes, using its T cell receptor, a boundcomplex between an MHC molecule (also referred to as an HLA molecule incase of human) on the cell surface of an antigen-presenting cell such asa dendritic cell or macrophage and an antigen peptide which is afragment peptide of an antigen protein derived from a cancer or virus,and thereby differentiates and proliferates. Antigen peptides presentedon the MHC molecules are known to be usually about 8 to 20 amino acidsin length. Antigen-specific T cells that thus have differentiated andproliferated exert their anti-tumor or anti-viral effects byspecifically injuring cancerous or virus-infected cells that present thecomplex bound between the antigen peptide and the MHC molecule, or byproducing various cytokines.

So-called vaccine therapies in which an antigen protein or an antigenpeptide derived from a cancer or virus is administered to potentiateantigen-specific T cells are believed useful for treatment or preventionof cancers and viral infections. Cancerous or viral antigens recognizedby T cells have been screened to date for various cancers and virusinfections, and many cancer antigen proteins, virus-derived antigenproteins, and antigen peptides derived therefrom have been alreadyidentified (Immunogenetics 1995, 41:178; Cancer Immunol. Immunother.2001, 50:3). For example, one of those antigens, WT1, was originallyidentified as a causative gene for a childhood renal tumor, that is,Wilms tumor (Nature 1990, 343:774). In normal tissues, the WT1 gene isweakly expressed only in restricted tissues such as kidney, testis, andovary, whereas it has been shown to be highly expressed in variouscancers such as leukemia as well as lung, breast, ovarian, prostatic,bladder, uterine, cervical, gastric, colon, germ cell, hepatic, and skincancers (JP Kokai H09-104627, JP Kokai H11-35484). Recently, it has beenshown that WT1-specific cytotoxic T cells (CTLs) were induced by invitro stimulation of peripheral blood mononuclear cells from HLA-A2.1-or HLA-A24.2-positive human donors with a 9-mer WT1 peptide comprisingan MHC class I binding motif (Immunogenetics 51:99-107, 2000; Blood95:2198-203, 2000; Blood 95:286-93, 2000). It has also been shown thatWT1-specific CTLs were induced by in vivo immunization of mice with a9-mer WT1 peptide (J Immunol 164:1873-80, 2000; Blood 96:1480-9, 2000)or WT1 cDNA (J Clin Immunol 20:195-202, 2000), and further that theimmunized mice reject transplanted tumor cells highly expressing WT1 (JImmunol 164:1873-80, 2000; J Clin Immunol 20:195-202, 2000). Thesefindings demonstrate that WT1 protein is one of cancer antigen proteins,and may provide a measure for cancer vaccines against fluid or solidcancers.

In order to efficiently induce a specific immunity by vaccination, it iseffective to administer an antigen protein or an antigen peptide as aprincipal agent in combination with a non-specific immunopotentiator.Known non-specific immunopotentiators include bacterium-derivedcomponents, cytokines, plant-derived components, and marineorganism-derived components. Bacterium-derived components include a deadbody of the BCG strain of Mycobacterium bovis, a cell wall skeletonintegrant of said BCG strain (BCG-CWS), a human tuberclebacillus-derived polysaccharide material (e.g. Ancer), a hemolyticstreptococcus powders (e.g. Picibanil), a bacterium-derivedpolysaccharide (e.g. lentinan, Krestin), a dead microbial suspensioncocktail (e.g. Broncasma Berna), a muramyldipeptide (MDP)-relatedcompound, a lipopolysaccharide (LPS), a lipid A-related compound (MPL),a glycolipid trehalose dimycolate (TDM), and a DNA derived from thebacteria as mentioned above (e.g. CpG oligonucleotides). Among them, aBCG-CWS has been known to exhibit an effective immunopotentiating actionwhen dispersed in an oil such as mineral oils (Cancer Res., 33,2187-2195 (1973); J. Nat. Cancer Inst., 48, 831-835 (1972); J.Bacteriol., 94, 1736-1745 (1967); Gann, 69, 619-626 (1978); J.Bacteriol., 92, 869-879 (1966)).

In addition, the dosage form of vaccine is also an important factor forefficient induction of specific immunity. For example, aluminiumpreparations, lipid particles, emulsion preparations, and microspheresare known as dosage forms of vaccines.

These substances and dosage forms that effect the enhancement of vaccineefficacies are collectively called adjuvants (Nature Biotech. 1999,17:1075). At present, the most widely used adjuvant among those asapproved for human use is an aluminium preparation, but its ability toinduce antigen-specific T cells is low, and side effects such as IgEproduction have been pointed out as problems.

In the light of the great ability of dendritic cells asantigen-presenting cells to induce antigen-specific T cells, research oncell vaccines has been also conducted in recent years, in whichdendritic cells derived from a patient are pulsed in vitro with anantigen protein or an antigen peptide to cause antigen presentation, andthen put back into the patient (Nature Med. 1998, 4:328). However, thereare many problems to be solved before the cell vaccine therapy is widelyavailable; it is technically difficult and costly to obtain a largeamount of dendritic cells required for the therapy.

Under such circumstances, there has been a need for developing a novelvaccine that enables simple, convenient, and efficient induction ofantigen-specific T cells as well as a method of administering the same.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a novel method by whichantigen-specific T cells can be efficiently induced. The object is thusto provide a method of inducing antigen-specific T cells which comprisesadministering a composition comprising a non-specific immunopotentiator,BCG-CWS, in advance, and then administering a composition comprising anantigen protein or an antigen peptide. A further object of the presentinvention is to provide a composition for treating and/or preventingcancers that is characterized in that it comprises a cancer antigenprotein, WT1, or a cancer antigen peptide derived from said WT1 proteinin combination with a BCG-CWS.

In order to elicit immune responses to a vaccine, it is important totimely administer an antigen in combination with an appropriatenon-specific immunopotentiator. As described above, however, there hasnot been known any method of administering a vaccine that efficientlyinduces antigen-specific T cells and thereby produces anti-tumor oranti-viral effects.

The present inventors therefore concentrated their efforts on examiningtherapeutic effects on an in vivo cancer model, of the use of cancerantigen peptides derived from the cancer antigen protein WT1, as used asan example, in combination with a non-specific immunopotentiator,BCG-CWS. As a result, it was found for the first time that a drasticimprovement in the induction of antigen-specific T cells, leading forexample to anti-tumor effects, can be obtained by a novel approachwherein a BCG-CWS is administered in advance and, after a certainperiod, an antigen peptide is administered, compared to the approacheswherein an antigen or a BCG-CWS is solely administered. The inventorsbelieve that this administration method should provide a similar effectwhatever antigen protein or antigen peptide is combined with a BCG-CWS.

Furthermore, in the present invention, the cancer antigen protein WT1was, for the first time, examined for its effects in a cancer model thatreflects, so to speak, “a therapeutic system in which a cancer antigenpeptide derived from WT1 is administered after the transplantation oftumor cells into an animal. As a result, it was demonstrated for thefirst time that WT1 is therapeutically effective. In addition, it wasfound that cancer antigen peptides derived from said WT1 protein produceremarkable anti-tumor effects when administered in combination with aBCG-CWS.

The present invention is based on such findings as described above.

Thus, the present invention relates to:

-   (1) a method for inducing antigen-specific T cells in a patient    comprising administering to said patient in need thereof;    composition (a) which comprises a therapeutically effective amount    of an antigen protein or an antigen peptide as an active ingredient,    and composition (b) which comprises a therapeutically effective    amount of a cell wall skeleton integrant of the BCG strain of    Mycobacterium bovis (BCG-CWS) as an active ingredient, wherein    composition (b) is administered in advance and then composition (a)    is administered;-   (2) A method according to above (1), wherein composition (a) is    administered about 24 hours after the administration of composition    (b);-   (3) A method according to above (1) or (2), wherein compositions (a)    and (b) are both administered intradermally;-   (4) A method according to above (3), wherein compositions (a)    and (b) are both administered intradermally at the same site;-   (5) A method according to any one of above (1) to (4), wherein the    administration cycle involving compositions (a) and (b) is repeated    two or more times;-   (6) A method according to any one of above (1) to (5), wherein    composition (a) comprises a cancer antigen protein or a cancer    antigen peptide as an active ingredient;-   (7) A method according to above (6), wherein the cancer antigen    protein or the cancer antigen peptide is WT1 protein set forth in    SEQ ID NO: 1 or a cancer antigen peptide derived from said WT1    protein;-   (8) A method according to above (7), wherein the cancer antigen    peptide derived from WT1 protein is selected from the group    consisting of Arg Met Phe Pro Asn Ala Pro Tyr Leu (SEQ ID NO: 2),    Cys Met Thr Trp Asn Glen Met Asn Leu (SEQ ID NO: 3), and Cys Tyr Thr    Trp Asn Gln Met Asn Leu (SEQ ID NO: 4);-   (9) A method of treatment and/or prevention of a cancer in a patient    which comprises a method according to any one of above (1) to (8);    and in another embodiment,-   (10) A pharmaceutical composition for enhancing an activity of an    antigen protein or an antigen peptide to induce antigen-specific T    cells, which comprises a cell wall skeleton integrant of the BCG    strain of Mycobacterium bovis as an active ingredient, and which is    administered before the administration of the antigen protein or the    antigen peptide;-   (11) A pharmaceutical composition for enhancing an anticancer    activity based on the immunopotentiating action of a cell wall    skeleton integrant of the BCG strain of Mycobacterium bovis, which    comprises an antigen protein or an antigen peptide as an active    ingredient, of which the activity to induce antigen-specific T cells    facilitates the enhancement of the anticancer activity, said    composition being administered after the administration of the cell    wall skeleton integrant of the BCG strain of Mycobacterium bovis;-   (12) A pharmaceutical composition according to above (10) or (11),    wherein the antigen protein or the antigen peptide is a cancer    antigen protein or a cancer antigen peptide;-   (13) A pharmaceutical composition according to above (12), wherein    the cancer antigen protein or the cancer antigen peptide is WT1    protein set forth in SEQ ID NO: 1 or a cancer antigen peptide    derived from said WT1 protein;-   (14) A pharmaceutical composition according to claim 13, wherein the    cancer antigen peptide derived from WT1 is selected from the group    consisting of Arg Met Phe Pro Asn Ala Pro Tyr Leu (SEQ ID NO: 2),    Cys Met Thr Trp Asn Gln Met Asn Leu (SEQ ID NO: 3), and Cys Tyr Thr    Trp Asn Gln Met Asn Leu (SEQ ID NO: 4);-   (15) A pharmaceutical composition according to any one of above (10)    to (14) for treatment and/or prevention of a cancer; and

in relation to the embodiment as mentioned above,

-   (16) A use of a cell wall skeleton integrant of the BCG strain of    Mycobacterium bovis for preparing a medicament which is administered    before the administration of an antigen protein or an antigen    peptide and which enhances an activity of the antigen protein or the    antigen peptide to induce antigen-specific T cells;-   (17) A use of an antigen protein or an antigen peptide for preparing    a medicament which is administered after the administration of a    cell wall skeleton integrant of the BCG strain of Mycobacterium    bovis and which enhances an anticancer activity based on the    immunopotentiating action of said a cell wall skeleton integrant of    the BCG strain of Mycobacterium bovis, wherein the anticancer    activity is enhanced by the activity of the cancer antigen protein    or the cancer antigen peptide to induce antigen-specific T cells;    and a use corresponding to the embodiments according to above (12)    to (15); and

in another embodiment,

-   (18) A pharmaceutical composition for enhancing an activity of WT1    protein set forth in SEQ ID NO: 1 or a cancer antigen peptide    derived from said WT1 protein to induce antigen-specific T cells,    which comprises a cell wall skeleton integrant of the BCG strain of    Mycobacterium bovis (BCG-CWS) as an active ingredient;-   (19) A pharmaceutical composition for enhancing an anticancer    activity based on the immunopotentiating action of a cell wall    skeleton integrant of the BCG strain of Mycobacterium bovis    (BCG-CWS), which comprises as an active ingredient WT1 protein set    forth in SEQ ID NO: 1 or a cancer antigen peptide derived from said    WT1 protein, of which the activity to induce antigen-specific T    cells facilitates the enhancement of the anticancer activity;-   (20) A pharmaceutical composition according to above (18) or (19),    wherein the cancer antigen peptide derived from WT1 is selected from    the group consisting of Arg Met Phe Pro Asn Ala Pro Tyr Leu (SEQ ID    NO: 2), Cys Met Thr Trp Asn Gln Met Asn Leu (SEQ ID NO: 3), and Cys    Tyr Thr Trp Asn Gln Met Asn Leu (SEQ ID NO: 4);-   (21) A pharmaceutical composition according to any one of above (18)    to (20) for treatment and/or prevention of a cancer; and

in relation to the embodiment as mentioned above,

-   (22) A method for enhancing, in a patient, an activity of WT1    protein set forth in SEQ ID NO: 1 or a cancer antigen peptide    derived from said WT1 protein to induce antigen-specific T cells,    which comprises administering to the patient a cell wall skeleton    integrant of the BCG strain of Mycobacterium bovis in an amount    effective to enhance the activity to induce the antigen-specific T    cells;-   (23) A method for enhancing, in a patient, an anticancer activity    based on the immunopotentiating action of a cell wall skeleton    integrant of the BCG strain of Mycobacterium bovis, which comprises    administering to the patient WT1 protein set forth in SEQ ID NO: 1    or a cancer antigen peptide derived from said WT1 protein in an    amount effective to enhance the anticancer activity, wherein the    anticancer activity is enhanced by the activity of the cancer    antigen protein or the cancer antigen peptide to induce    antigen-specific T cells;-   (24) A method according to above (22) or (23), wherein the cancer    antigen peptide derived from WT1 protein is selected from the group    consisting of Arg Met Phe Pro Asn Ala Pro Tyr Leu (SEQ ID NO: 2),    Cys Met Thr Trp Asn Gln Met Asn Leu (SEQ ID NO: 3), and Cys Tyr Thr    Trp Asn Gln Met Asn Leu (SEQ ID NO: 4);-   (25) A method according to any one of above (22) to (24) for    treatment and/or prevention of a cancer;-   (26) A use of a cell wall skeleton integrant of the BCG strain of    Mycobacterium bovis for preparing a medicament which enhances an    activity of WT1 protein set forth in SEQ ID NO: 1 or a cancer    antigen peptide derived from said WT1 protein to induce    antigen-specific T cells;-   (27) A use of WT1 protein set forth in SEQ ID NO: 1 or a cancer    antigen peptide derived from WT1 protein for preparing a medicament    which enhances an anticancer activity based on the    immunopotentiating action of a cell wall skeleton integrant of the    BCG strain of Mycobacterium bovis, wherein the anticancer activity    is enhanced by the activity of the cancer antigen protein or the    cancer antigen peptide to induce antigen-specific T cells;-   (28) A use according to above (26) or (27), wherein the cancer    antigen peptide derived from WT1 protein is selected from the group    consisting of Arg Met Phe Pro Asn Ala Pro Tyr Leu (SEQ ID NO: 2),    Cys Met Thr Trp Asn Gln Met Asn Leu (SEQ ID NO: 3), and Cys Tyr Thr    Trp Asn Gln Met Asn Leu (SEQ ID NO: 4); and-   (29) A use according to any one of above (26) to (28) for preparing    a medicament for treatment and/or prevention of a cancer.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a schedule for transplantation of tumor cells andvaccination with a WT1 peptide and a BCG-CWS.

FIG. 2 is a graph showing the longer diameters of tumors in mm up untilDay 65 after transplantation of tumor cells on vaccinations. In thefigure, solid circles indicate results of WT1 peptide+BCG-CWSadministration, open triangles indicate results of WT1 peptideadministration, solid diamonds indicate results of BCG-CWSadministration, and solid squares indicate results without anyvaccination.

FIG. 3 is a graph showing the survival ratio (%) of mice up until Day 65after transplantation of tumor cells on vaccinations. In the figure, —indicates the results WT1 peptide+BCG-CWS administration, —•—• indicatesthe result of WT1 peptide administration, - - - indicates the result ofBCG-CWS administration, and • • • indicates the result without anyvaccination.

FIG. 4 is a graph showing the disease free survival rate (%) of mice upuntil Day 65 after transplantation of tumor cells on vaccinations. Inthe figure, — indicates the results of WT1 peptide+BCG-CWSadministration, —•—• indicates the result of WT1 peptideadministration, - - - indicates the result of BCG-CWS administration,and • • • indicates the result without any vaccination.

FIG. 5 is a graph showing the results of colony assay of the myeloidcells removed on Day 65 from the mice transplanted with tumor cells andvaccinated. In the figure, solid bars indicate results of WT1peptide+BCG-CWS administration, stippled bars indicate results ofBCG-CWS administration, and open bars indicate results without anyvaccination.

FIG. 6 is a graph showing the cytotoxic effects of splenocytes from themice that any tumor has not been established by coadministration of WT1peptide and BCG-CWS and from the untreated mice on WT1-C1498 cells andC1498 cells.

BEST MODE FOR CARRYING OUT THE INVENTION

As described above, according to a first aspect, the present inventionprovides a method for inducing antigen-specific T cells in a patientcomprising administering to said patient in need thereof composition (a)which comprises a therapeutically effective amount of an antigen proteinor an antigen peptide as an active ingredient and composition (b) whichcomprises a therapeutically effective amount of a cell wall skeletonintegrant of the BCG strain of Mycobacterium bovis as an activeingredient, wherein said composition (b) is administered in advance andthen said composition (a) is administered. This method is characterizedin that it comprises an administration procedure in which the BCG-CWS asan adjuvant is administered in advance, and then, after a certainperiod, an antigen (an antigen protein or antigen peptide derived fromsaid antigen protein) is administered. The administration procedure ofthe present invention can drastically improve an anti-tumor effect,antiviral effect, and disease free survival rate, compared to theadministration of the antigen alone, or the BCG-CWS alone.

The term “antigen protein or antigen peptide” comprised as an activeingredient in composition (a) refers to an antigen protein and anantigen peptide derived from said antigen protein, and is notspecifically limited so long as it is capable of inducing T cellsspecific for an antigen peptide. In addition, the term “antigen proteinor antigen peptide” also includes within its scope both those capable ofinducing antigen-specific T cells directly by forming a complex with anMHC molecule (HLA molecule) on the cell surface of an antigen-presentingcell as well as those capable of inducing antigen-specific T cellsindirectly, that is, by being incorporated into the cell andintracellularly degraded to a peptide fragment which in turn binds anMHC molecule to form a complex presented on the cell surface.

Antigen proteins include, for example, antigen proteins derived fromviruses, antigen proteins derived from bacteria, or cancer antigenproteins (also referred to as tumor antigen proteins). Several proteinsalready known as antigen proteins are listed below. Examples of antigenproteins derived from viruses are those derived from HIV, hepatitis Cvirus, hepatitis B virus, influenza virus, HPV, HTLV, and EBV. Examplesof antigen proteins derived from bacteria are those derived fromtubercle bacilli. Representative examples of cancer antigen proteins arethose listed in Table 1 of Immunity, vol. 10: 281, 1999, or those listedin Tables 1 to 6 of Cancer Immunol. Immunother., vol.50, 3-15, 2001.More specifically, melanoma antigen proteins include MAGE (Science,254:1643, 1991), gp100 (J. Exp. Med., 179:1005, 1994), MART-1 (Proc.Natl. Acad. Sci. USA, 91:3515, 1994), and tyrosinase (J. Exp. Med.,178:489, 1993); and cancer antigen proteins other than those derivedfrom melanoma includes tumor makers such as HER2/neu (J. Exp. Med.,181:2109, 1995), CEA (J. Natl. Cancer. Inst., 87:982, 1995), and PSA (J.Natl. Cancer. Inst., 89:293, 1997), as well as SART-1 derived fromsquamous cell carcinoma (J. Exp. Med., vol. 187, p. 277-288, 1998; WO97/46676), cyclophilin B (Proc. Natl. Acad. Sci., U.S.A. 88:1903, 1991),SART-3 (Cancer Res., vol. 59, 4056 (1999)), and WT1 (Immunogenetics,vol. 51, 99, 2000; Blood 95:2198-203, 2000; Blood 95: 286-93, 2000, orhuman WT1 set forth in the Sequence Listing of the present applicationas SEQ ID NO: 1). In addition to the above antigen proteins in theirfull-length forms, partial polypeptides or alterations thereof are alsoincluded so long as they are capable of inducing T cells specific for anantigen peptide.

Such antigen proteins may be obtained through the following steps:cloning a cDNA encoding a desired antigen protein, ligating the cDNAinto an expression vector, introducing the resulting recombinantexpression vector into a host cell, and expressing the antigen protein,according to the references cited above or to standard texts such asMolecular Cloning 2nd Ed., Cold Spring Harbor Laboratory Press (1989).More specifically, for example, a cDNA encoding a desired antigenprotein is cloned by hybridization or a PCR method. The cloned cDNA isthen incorporated into an appropriate expression vector (e.g.pSV-SPORT1). The resulting recombinant expression vector is introducedinto a host cell, and the transformants thus obtained may be cultured inan appropriate medium to express and produce the desired antigenprotein. In this context, host cells include, for example, prokaryotessuch as E. coli, unicellular eukaryotes such as yeasts, andmulticellular eukaryotic cells such as those of insects or animals.Methods of gene transfer into host cells include, for example, thecalcium phosphate, DEAE-dextran, and electric pulse methods.Polypeptides thus obtained can be isolated and purified using standardbiochemical techniques.

The in vitro ability of such antigen proteins to induce antigen-specificT cells can be examined, for example, in the case of cancer antigenproteins, by a test as described below. Thus, first, certain cells whichexpress no cancer antigen protein such as COS-7 derived from Africangreen monkey kidney (ATCC CRL1651) or fibroblast VA-13 (RIKEN Cell Bank,The Institute of Physical and Chemical Research) are double transfectedwith a recombinant expression vector which comprises cDNA encoding adesired cancer antigen protein and a recombinant expression vector whichcomprises DNA encoding an HLA antigen. Such transfection may be achievedby the Lipofectin method using Lipofectamine reagent (GIBCO BRL).Subsequently, tumor-reactive CTLs restricted by the HLA molecule usedare added and allowed to act. The amounts of various cytokines (e.g.IFN-γ) produced by said CTLs in response may be then measured, forexample, by an ELISA method, in order to evaluate an activity of thedesired cancer antigen protein to induce antigen-specific T cells.

Antigen peptides derived from antigen proteins (hereinafter simplyreferred to as antigen peptides) include, for example, peptides of about8 to 20 amino acid residues which are part of said antigen proteins, oraltered peptides thereof having functionally equivalent properties, orpolytopes in which two or more of said peptides or altered peptidethereof are linked together. In this definition, the range “8 to 20” isbased on the common knowledge among those skilled in the art thatantigen peptides presented by MHC molecules are usually about 8 to 20amino acids in length. The term “altered peptide having functionallyequivalent properties” means an altered peptide in which one to severalamino acid residues in the amino acid sequence of an antigen peptidehave been substituted, deleted, and/or added (including addition to theamino acid at the N- or C-terminal end of the peptide) and which iscapable of inducing T cells specific for an antigen peptide.

In cancer antigen peptides and virus-derived antigen peptides, certainrules (motifs) in the sequences of antigen peptides bound and presentedby an HLA molecule are known for certain HLA types such as HLA-A1,-A0201, -A0204, -A0205, -A0206, -A0207, -A11, -A24, -A31, -A6801, -B7,-B8, -B2705, -B37, -Cw0401, and -Cw0602 (see, e.g., Immunogenetics,41:178, 1995). For example, regarding a motif for HLA-A24, it is knownthat the amino acid at position 2 in a peptide consisting of 8 to 11amino acids is tyrosine, phenylalanine, methionine, or tryptophan, andthe amino acid at the C-terminal end is phenylalanine, leucine,isoleucine, tryptophan, or methionine (J. Immunol., 152, p. 3913, 1994;Immunogenetics, 41, p. 178, 1995; J. Immunol., 155, p. 4307, 1994).Likewise, regarding HLA-A2, motifs listed below in Table 1 are known(Immunogenetics, 41, p. 178, 1995; J. Immunol., 155, p. 4749, 1995).TABLE 1 Amino acid at position 2 Amino acid at the C- Type of HLA-A2from the N-terminal end terminal end HLA-A0201 L, M V, L HLA-A0204 L LHLA-A0205 V, L, I, M L HLA-A0206 V, Q V, L HLA-A0207 L L(the peptides are 8 to 11 amino acids in length)

Furthermore, in recent years, peptide sequences expected to be able tobind HLA antigens can be searched on the Internet by using a software ofBIMAS at NIH (http://bimas.dcrt.nih.gov/molbio/hla_bind/). Such peptidesequences also can be searched by using BIMAS HLA peptide bindingprediction analysis (J. Immunol., 152, 163, 1994).

It is therefore easy to select antigen peptide portions involved inthese motifs from the amino acid sequences of the cancer antigenproteins or virus-derived antigen proteins as described above. Specificexamples of antigen peptides thus selected, in particular, for example,of cancer antigen peptides, are as follows. Examples of cancer antigenpeptides derived from WT1 are peptides listed in Table II to Table XLVIof WO 2000/18795, and in particular, peptides having the HLA-A24 andHLA-A2 binding motifs set forth in the Sequence Listing of the presentapplication as SEQ ID NOs: 2 and 3. Examples of cancer antigen peptidesderived from SART-1 are peptides listed in the Sequence Listings of WO97/46676, WO 2000/02907, and WO 2000/06595. Examples of cancer antigenpeptides derived from cyclophilin B are peptides listed in the SequenceListing of WO 99/67288. Examples of cancer antigen peptides derived fromSART-3 are peptides listed in the Sequence Listing of WO 2000/12701. Bysubjecting the above peptides to an activity measurement describedbelow, one can select antigen peptides having an activity to induceantigen-specific T cells.

In addition, where certain rules (motifs) in the antigen peptidesequences that are bound and presented by an HLA molecule are known asdescribed above, altered peptides having properties functionallyequivalent to those of the above antigen peptides may be exemplified byaltered peptides in which one or more of amino acids have beensubstituted on the basis of said motifs. Thus, in the case of a bindingmotif for HLA-A24, for example, it is known as described above that theamino acid at position 2 in a peptide consisting of 8 to 11 amino acidsis tyrosine, phenylalanine, methionine, or tryptophan, and the aminoacid at the C-terminal end is phenylalanine, leucine, isoleucine,tryptophan, or methionine (J. Immunol., 152, p. 3913, 1994;Immunogenetics, 41, p. 178, 1995; J. Immunol., 155, p. 4307, 1994).Therefore, altered peptides bound and presented by the HLA-A24 antigenmay be exemplified by those in which one or more amino acids at position2 and at the C-terminal end of an HLA-A24-restricted wild-type peptidehave been substituted within the amino acids listed above. Specificexamples of such altered peptides, for example, in connection withcancer antigens, are as follows. Examples of altered peptides derivedfrom WT1 are those obtained by modifying the peptides listed in Table IIto Table XLVI of WO2000/18795 on the basis of the above motif, and inparticular, a peptide having the amino acid sequence set forth in theSequence Listing of the present application as SEQ ID NO: 4. Likewise,examples of altered peptides derived from, for example, SART-1,cyclophilin B, or SART-3 are those obtained by modifying respectiveantigen peptides disclosed in the above references on the basis of themotif.

Antigen peptides (including altered peptides) as described above may beprepared according to the methods usually used in peptide chemistry.Examples of such methods are those described in references, for example,“Peptide Synthesis”, Interscience, New York, 1966; “The Proteins”, Vol.2, Academic Press Inc., New York, 1976; “Peputido-Gosei”, Maruzen, 1975;“Pepuchido-Gosei-no-Kiso-to-Jikken”, Maruzen, 1985; and“Iyakuhin-no-Kaihatu, Zoku, Vol. 14, Peputido-Gosei”, Hirokawa Shoten,1991. Alternatively, such peptides may also be prepared according to“Molecular Cloning” cited above by expressing a recombinant peptide froma DNA encoding the antigen peptide and purifying it using routineprocedures.

The in vitro ability of such antigen peptides to induce antigen-specificT cells can be examined, for example, in the case of cancer antigenpeptides, by an assay described, for example, in J. Immunol., 154, p.2257, 1995. In particular, peripheral blood lymphocytes may be isolatedfrom an HLA antigen-positive human, and stimulated in vitro by adding apeptide of interest. If CTLs that specifically recognize theHLA-positive cells pulsed with the peptide are induced, the peptide canbe thereby confirmed to have an activity to induce antigen-specific Tcells. The presence or absence of CTL induction may be determined, forexample, by measuring the amount of IFN-γ produced by CTLs in responseto the antigen peptide-presenting cells using an enzyme-linkedimmunosorbent assay (ELISA). Alternatively, the amount of TNF-α producedby CTLs in response to the antigen peptide-presenting cells may bedetermined by measuring the survival rate of a TNF-α sensitive cell line(e.g. WEHI164S cells; ATCC Cat. No. CRL-1751).

Determination may also be achieved by a method in which the cytotoxicityof CTLs against antigen peptide-presenting cells labeled with ⁵¹Cr ismeasured (⁵¹Cr release assay; Int. J. Cancer, 58:317, 1994).Alternatively, for example, COS-7 (ATCC No. CRL1651) or VA-13 (RIKENCell Bank, The Institute of Physical and Chemical Research) cells intowhich an expression plasmid expressing cDNA for HLA has been introducedare pulsed with a peptide of interest. Then, for example, the CTLsprepared as described above may be reacted with the pulsed cells, andthe amounts of various cytokines (e.g. IFN-γ or TNF-α) produced by saidCTLs may be measured.

The term “polytope” means a recombinant peptide in which two or moreantigen peptides have been linked together (see, e.g. , Journal ofImmunology, 160, p. 1717, 1998), and particularly in the presentinvention refers to a polypeptide which appropriately combines one, twoor more kinds of the above antigen peptides. A polytope is obtained by aprocedure in which a recombinant DNA prepared by linking together one,two or more kinds of DNAs encoding the above antigen peptides isinserted into an appropriate expression vector, and the recombinantvector obtained is then expressed in a host cell. Activity of thepolytope to induce antigen-specific T cells may be confirmed bysubjecting it to the assay for antigen protein as described above.

At least one kind of antigen protein or peptide as described above isselected and used as an active ingredient in the above composition (a).Depending on the purpose, two or more kinds of antigen proteins orpeptides may be present. Although a therapeutically effective amount ofsuch antigen proteins or peptides is not specifically limited so long asit is capable of inducing in vivo antigen-specific T cells, it ispreferably usually 0.0001 mg to 1000 mg, more preferably 0.001 mg to 100mg, still more preferably 0.01 mg to 10 mg.

The above composition (a) is preferably formulated into a dosage formthat achieves desired pharmacological effects. Dosage forms suitable forthis purpose include, for example, formulations such as water-in-oil(w/o) emulsions, oil-in-water (o/w) emulsions, and water-in-oil-in-water(w/o/w) emulsions, as well as liposome formulations, microsphereformulations, microcapsule formulations, solid injections and liquidformulations.

Water-in-oil (w/o) emulsion formulations take the form in which anactive ingredient is dispersed in an aqueous dispersed phase.Oil-in-water (o/w) emulsion formulations take the form in which anactive ingredient is dispersed in an aqueous dispersion medium.Likewise, water-in-oil-in-water (w/o/w) emulsion formulations take theform in which an active ingredient is dispersed in the inner-mostaqueous dispersed phase. Preparation of such formulations may beachieved by referring to, for example, JP Kokai H08-985, JP KokaiH09-122476, etc.

Liposome formulations comprise microparticles in the form in which anactive ingredient is incorporated in an aqueous phase or withinmembranes by means of liposomes having a lipid bilayer structure.Examples of principal lipids for preparing liposomes arephosphatidylcholine and sphingomyelin, to which, for example, dicetylphosphate, phosphatidic acid, or phosphatidylserine is added to provideliposomes with electric charges for stabilization. Exemplary methods forpreparing liposomes are ultrasonication, ethanol injection, etherinjection, reverse-phase evaporation, and French press extractionmethods.

Microsphere formulations comprise microparticles made of a homogeneouspolymer matrix in which an active ingredient is dispersed. Examples ofcomponents for the matrix are biodegradable polymers such as albumin,gelatin, chitin, chitosan, starch, polylactic acid, and polyalkylcyanoacrylate. Preparation of microsphere formulations may be carriedout according to known methods (Eur. J. Pharm. Biopharm. 50:129-146,2000; Dev. Biol. Stand. 92:63-78, 1998; Pharm. Biotechnol. 10:1-43,1997) and is not specifically limited.

Microcapsule formulations comprise microparticles in the form in whichan active ingredient as a core substance is covered with anencapsulating substance. Examples of a coating material used as anencapsulating substance are membrane-forming polymers such ascarboxymethylcellulose, cellulose acetate phthalate, ethylcellulose,gelatin, gelatin-acacia, nitrocellulose, polyvinyl alcohol, andhydroxypropylcellulose. Microcapsule formulations may be preparedaccording to, for example, the coacervation or interfacialpolymerization method.

Solid injections are dosage forms in which an active ingredient isincluded in a base material such as collagen or silicone to solidify theforms. Solid injections may be prepared according to, for example, amethod described in a reference (Pharm. Tech. Japan, 7 (1991), p.402-409).

Liquid formulations are dosage forms in which an active ingredient ismixed with a pharmaceutically acceptable solvent, carrier, or the like.Examples of a pharmaceutically acceptable solvent include water, aglucose solution, and physiological saline. In addition, liquidformulations may comprise a pharmaceutically acceptable auxiliary agentsuch as a pH regulating agent or buffer, a tonicity adjusting agent, ora swelling agent.

Furthermore, composition (a) may also take the form of a lyophilizedformulation corresponding to the above dosage forms. Other agents suchas a stabilizing agent (e.g. polysaccharides, amino acids, proteins,urea, or sodium chloride), an excipient (e.g. sugars, amino acids, urea,or sodium chloride), an antioxidant, an antiseptic, an isotonizingagent, or an buffer may also be added if necessary.

Such composition (a) as described above may be used as a pre-formulatedproduct or may be prepared before use for administration to a patient.Thus, the antigen protein or the antigen peptide as an active ingredientof composition (a) as well as an emulsion or other preparation as thedosage form may be used as a pre-formed product in which theconstituents have already been mixed together, or may be prepared beforeuse for administration to a patient.

Composition (b), that is a composition comprising as an activeingredient a BCG-CWS is described below.

BCG-CWS that is a CWS of the BCG strain of Mycobacterium bovis may beisolated and prepared according to known literatures such as CancerRes., 33, 2187-2195 (1973), J. Natl. Cancer Inst., 48, 831-835 (1972),J. Bacteriol., 94, 1736-1745 (1967), Gann, 69, 619-626 (1978), J.Bacteriol., 92, 869-879 (1966), and J. Natl. Cancer Inst., 52, 95-101(1974). In brief, those CWSs may be obtained in a form of insolubleresidue by a purification process which comprises crashing the cellswith a physical means, removing nucleic acids and proteins from the celldebris, and then delipidating the resultant material.

Said BCG-CWS is preferably formulated into a dosage form that achieves adesired pharmacological effect. In order to attain the purpose, anemulsion is preferable, and an oil-in-water (o/w) emulsion is morepreferable. Oils as a constituent component of the oil-in-water emulsioninclude mineral oils or animal and vegetable oils as described inImmunology, 27, 311-329 (1974). The mineral oil is exemplified by aliquid petrolatum, a bayol (Bayol F), Drakeol-6VR, and the like. Thevegetable oil is exemplified by a peanut oil, a sesame oil, AD-65 (amixture of a peanut oil, Arlacel, and aluminum monostearate), and thelike. The animal oil is exemplified by squalane, and a terpenoidderivative such as squalene, and the like. Among them, Drakeol-6VR andsqualane are preferred.

BCG-CWS is preferably comprised in the oil-in-water emulsions in aconcentration range of 0.1 to 10 mg/ml. Oil is suitably comprised in aconcentration range of 0.01 to 30% w/w, preferably 0.01 to 10% w/w, andmore preferably 0.01 to 5.0% w/w.

Oil-in-water emulsion formulations may comprise a surfactant, astabilizer, an excipient or the like, if necessary. In this context, asurfactant is not limited to a particular species as long as it may beused in a pharmaceutical formulation. It includes a phospholipid, anonionic surfactant, and the like. Specific examples includephospholipids such as egg-yolk phosphatidyl amine, egg-yolk lecithin,soybean lecithin, nonionic surfactants such as polyoxyethylene sorbitanfatty acid esters, e.g. Polysorbate 80, and sorbitan fatty acid esters,e.g. Span 40. Each surfactant may be used solely, or in combination withany other several ones, if necessary.

Said stabilizers include a polysaccharide, an amino acid, a protein,urea, a sugar alcohol, and sodium chloride. Specific examples includepolysaccharides such as dextran, starch, cellulose, and amino acids suchas a neutral amino acid, e.g. alanine, glycine and proline. Proteinsinclude albumin, a gelatin, and a collagen. Sugar alcohols includemannitol and sorbitol. Each stabilizer may be used solely, or incombination with any other several ones, if necessary.

Said excipients include a saccharide, an amino acid, urea, and sodiumchloride. Specific examples include saccharides such as amonosaccharide, a disaccharide, and a sugar alcohol. Monosaccharidesinclude glucose, and fructose, and disaccharides include maltose,lactose, and trehalose. Sugar alcohols include mannitol, and sorbitol.Amino acids includes alanine, and glycine. Each excipient may be usedsolely, or in combination with any other several ones, if necessary.

In addition, an antioxidant, an antiseptic, an isotonizing agent, or anbuffer, each of which may be used in pharmaceutical formulations may becomprised, if necessary.

Oil-in-water emulsion formulations that comprise BCG-CWS as describedabove may also take the form of a lyophilized formulation. A dispersionsolvent to be used to re-disperse the lyophilized formulation is amedium for dispersing emulsion particles, and includes injectable water(injectable distilled water), a physiological saline and the like, butis limited to a particular species as long as it can be injected as adispersion solvent.

Details for the processes of preparation of such an oil-in-wateremulsion formulation that comprises BCG-CWS as described above arediscussed in WO00/3724. Specifically, the emulsion may be prepared by,for example, adding a BCG-CWS to an oil as described above, addingfurther an aqueous solution of a surfactant, an excipient, a stabilizerand another additive, and then emulsifying the resultant mixture with adispersing or emulsifying device such as a Potter-Elvehjem typehomogenizer, a homomixer, an ultrasonic homogenizer, Microfluidizer, orthe like, followed by lyophilizing the oil-in-water emulsion to givefinally a lyophilized formulation.

Such composition that comprises a BCG-CWS as an active ingredient incomposition (b) as prepared as shown above may be used as apre-formulated product, or may be prepared before use for administrationto a patient. Therapeutically effective amount of BCG-CWS is not limitedto specific one as long as it facilitates the enhancement of theactivity to induce antigen-specific T cells, and may be preferably 0.1to 200 μg, and more preferably 1 to 100 μg per administration.

A method of inducing antigen-specific T cells according to the presentinvention which comprises administering compositions (a) and (b) asdescribed above is characterized in that composition (b) is administeredin advance and then composition (a) is administered. In particular,composition (a) is preferably administered 6 or more hours after theadministration of composition (b), and more preferably composition (a)is administered 12 or more hours after the administration of composition(b). Further preferably, composition (a) is administered about 12 to 48hours after the administration of composition (b), and still morepreferably, composition (a) is administered about 24 to 48 hours afterthe administration of composition (b). The most preferable timing ofadministration is such that composition (a) is administered about 24hours (ca. 1 day; 20 to 28 hours) after the administration ofcomposition (b).

In this connection, administration may be carried out in any manner solong as the timing of administration is such that composition (b) isadministered in advance and then composition (a) is administered, asdescribed above, and examples of the administration procedure include:

-   1) composition (b) is administered one or more times, and after a    certain period as described above, composition (a) is administered;    or-   2) composition (b) is administered one or more times, and after a    certain period as described above, compositions (b) and (a) are    simultaneously administered.

In this regard, when composition (b) is administered two or more times,the number of administrations may be in particular 2 to 10, andpreferably 2 to 5.

Taking the above administrations of (a) and (b) as one administrationcycle, such an administration cycle may be repeated two or more times inorder to further improve the effect on induction of T cells. Thus, theadministration cycle may be repeated two or more times as appropriatedepending, for example, on the disease to be treated, the symptoms, age,and weight of the patient. The interval between the repeatedadministration cycles may also be determined as appropriate in the rangefrom about one week to about one year depending, for example, on thesymptoms of the patient.

The route of administration for compositions (a) and (b) used in amethod of inducing antigen-specific T cells according to the presentinvention may be, for example, intradermal administration, subcutaneousadministration, continuous subcutaneous administration, intravenousinjection, intraarterial injection, intramuscular injection, localinfusion, or intraperitoneal administration. It is also possible tocontinuously and slowly administer using, for example, an osmotic pump,or to prepare a sustained-release formulation (e.g. a mini-pelletformulation) and implant it. Preferred is intradermal or subcutaneousadministration. It is particularly preferred to intradermally administerboth of compositions (a) and (b). In that case, it is preferred tointradermally administer compositions (a) and (b) at the same site.

Exemplary combinations of the active ingredients of the abovecompositions (a) and (b) in a method of inducing antigen-specific Tcells according to the present invention may be combinations of anantigen protein with a BCG-CWS, or may also be combinations of anantigen peptide with a BCG-CWS. Among these combinations, when theantigen is a cancer antigen, specific examples include a combination ofWT1 protein (SEQ ID NO: 1) with a BCG-CWS and a combination of a cancerantigen peptide derived from WT1 with a BCG-CWS. In this context, thecancer antigen peptide derived from WT1 may be those peptides found inthe amino acid sequence of human WT1 set forth in SEQ ID NO: 1 whichhave a motif structure as described above that is bound and presented byan HLA antigen, as well as altered peptides thereof. Particular examplesare the peptides listed in Table II to Table XLVI of WO 2000/18795 andaltered peptides based on the motifs, and more preferable, Arg Met PhePro Asn Ala Pro Tyr Leu (SEQ ID NO: 2) and Cys Met Thr Trp Asn Gln MetAsn Leu (SEQ ID NO: 3) which have a binding motif for HLA-A2 andHLA-A24, or altered peptides thereof based on the HLA-A24 or HLA-A2binding motif. A specific example of such altered peptides is Cys TyrThr Trp Asn Gln Met Asn Leu (SEQ ID NO: 4) in which Met at position 2 ofthe peptide set forth in SEQ ID NO: 3 has been replace by Tyr, an aminoacid consistent with the motif.

A method of inducing antigen-specific T cells according to the presentinvention as described above may be examined for its ability to induceantigen-specific T cells as follows.

Composition (b) according to the present invention is injectedintradermally into a laboratory animal, and after 24 hours, composition(a) is injected intradermally. Taking these administrations as onecourse, vaccination is conducted once or several times at intervals ofone to two weeks. One week after the last administration, the spleen isremoved, and lymphocytes are prepared from the spleen. Splenocytes fromunprimed mice are also prepared in parallel, and pulsed with an antigenpeptide for several hours followed by X-irradiation at a dose of about2000 to 5000 rad to use as antigen-presenting cells. Lymphocytes fromimmunized mice are restimulated with the antigen peptide in a culturesystem by adding thereto the antigen-presenting cells. If necessary,similar stimulation is conducted several times at a frequency of once aweek. One week after the last stimulation, lymphocytes are recovered,and may be examined for their ability to induce antigen-specific T cellsusing target cells such as cells pulsed with the antigen peptide orcells positive for the antigen, for example, by determining the amountsof various cytokines (e.g. IFN-γ) produced in response by antigenpeptide-specific T cells induced among lymphocytes, or by measuring thecytotoxicity of antigen peptide-specific T cells against target cellslabeled with ⁵¹Cr according to the ⁵¹Cr release assay (J. Immunol.,139:2888, 1987). For human, peripheral blood mononuclear cells (PBMCs)isolated from peripheral blood, for example, by the Ficoll method may beused instead of splenic lymphocytes of laboratory animals in order toexamine, in a similar manner, the ability to induce antigenpeptide-specific T cells.

It is also possible to examine the ability to induce cancerantigen-specific T cells by the procedures described below in theExamples. Briefly, cDNA encoding a cancer antigen protein of interest isintroduced into a tumor cell to prepare tumor cells highly expressingthe cancer antigen protein of interest. The tumor cells are administeredintraperitoneally, and vaccination is started on the next day.Vaccination is achieved by four courses of administration conducted atintervals of one week; one course of administration consisting of firstintradermally injecting composition (b) as described above, and after 24hours, intradermally injecting composition (a). Then, anti-tumor effectsbased on the ability to induce antigen-specific T cells may be examinedby measuring a tumorigenic rate, a survival rate, or a disease-freesurvival rate using a routine method. Alternatively, an administrationprocedure similar to that described above may be conducted on a tumorpatient instead of a laboratory animal to examine the ability to induceantigen peptide-specific T cells.

When applied to an antigen-positive patient, a method of inducingantigen-specific T cells according to the present invention can cause anantigen peptide to be presented at a high density with an HLA antigen onantigen-presenting cells and can thereby induce proliferation of T cellsspecific for the presented HLA antigen-peptide complex, leading tokilling of target cells (cells positive for the antigen peptide) oractivation of immunity by production of various cytokines. A method ofinducing antigen-specific T cells according to the present invention,where the antigen is a cancer antigen, is used for treatment orprevention of a cancer. In particular, it is used for treatment orprevention of, for example, lung, ovarian, and prostatic cancer as wellas leukemia. Likewise, the method is used for treatment or prevention ofviral infections, where the antigen is a virus-derived antigen.

In treatment or prevention of a cancer, a method of inducingantigen-specific T cells according to the present invention can induceand enhance specific cellular immunity against cancer cells, and therebytreat a cancer or prevent proliferation and metastasis of a cancer. Inaddition, a method of inducing antigen-specific T cells according to thepresent invention may be used in combination with a conventionalchemotherapy or radiotherapy to enhance the therapeutic effects. Intreatment or prevention of a viral infection, a method of inducingantigen-specific T cells according to the present invention can induceand enhance specific cellular immunity against virus-infected cells, andcan thereby treat or prevent a virus infection.

Although the timing of starting a method of inducing antigen-specific Tcells according to the present invention is not specifically limited,the method may be preferably carried out, for example, after a patientwith leukemia has attained complete remission (CR), or during situationsin which the number of tumor cells has been reduced by a solid cancersurgery, that is, the patient has achieved a state of minimal residualdisease (MRD).

In connection with the above embodiments, the present invention providesa method of treatment and/or prevention of a cancer in a patientcomprising a method for inducing antigen-specific T cells in the patientaccording to the present invention.

In another embodiment, the present invention relates to a pharmaceuticalcomposition for enhancing an activity of an antigen protein or anantigen peptide to induce antigen-specific T cells, which comprises aBCG-CWS as an active ingredient, and which is administered before theadministration of the antigen protein or the antigen peptide; and

to a pharmaceutical composition for enhancing an anticancer activitybased on the immunopotentiating action of a BCG-CWS, which comprises anantigen protein or an antigen peptide as an active ingredient and whichis administered after the administration of said non-specificimmunopotentiator, wherein the anticancer activity is enhanced by theactivity to induce antigen-specific T cells. The compositions comprisinga BCG-CWS as described above may be prepared and used according to theabove descriptions for composition (b), while the compositionscomprising an antigen protein or an antigen peptide as described abovemay be prepared and used according to the above descriptions forcomposition (a).

In connection with the above embodiments, the present invention providesa use of a BCG-CWS for preparing a medicament which is administeredbefore the administration of an antigen protein or an antigen peptideand which enhances an activity of the antigen protein or the antigenpeptide to induce antigen-specific T cells, as well as a use of anantigen protein or an antigen peptide for preparing a medicament whichis administered after the administration of a BCG-CWS and which enhancesan anticancer activity based on the immunopotentiating action of saidBCG-CWS, wherein the anticancer activity is enhanced by the activity ofthe cancer antigen protein or the cancer antigen peptide to induceantigen-specific T cells.

In particular embodiments, the present invention also provides apharmaceutical composition for enhancing an activity of WT1 protein setforth in SEQ ID NO: 1 or a cancer antigen peptide derived from said WT1protein to induce antigen-specific T cells, which comprises a BCG-CWS asan active ingredient, as well as a pharmaceutical composition forenhancing an anticancer activity based on the immunopotentiating actionof a BCG-CWS, which comprises as an active ingredient WT1 protein setforth in SEQ ID NO: 1 or a cancer antigen peptide derived from said WT1protein wherein the anticancer activity is enhanced by the activity ofthe protein or the peptide to induce antigen-specific T cells. Thepresent embodiments encompass a pharmaceutical composition for enhancingan activity of WT1 protein set forth in SEQ ID NO: 1 or a cancer antigenpeptide derived from said WT1 protein to induce antigen-specific Tcells, which comprises WT1 protein set forth in SEQ ID NO: 1 or a cancerantigen peptide derived from said WT1 protein along with a BCG-CWS, anda pharmaceutical composition for enhancing an anticancer activity basedon the immunopotentiating action of a BCG-CWS, which comprises WT1protein set forth in SEQ ID NO: 1 or a cancer antigen peptide derivedfrom said WT1 protein along with a BCG-CWS.

As shown in Examples hereinafter, when used in combination with BCG-CWSfor the first time, WT1 has been demonstrated to have the effects on thetreatment of tumors and the effects on the improvement in disease freesurvival rate. The effects were much better than those on the approacheswherein an antigen or a BCG-CWS is solely administered. It is thereforeexpected that a therapeutic and/or prophylactic agent for cancer whichinvolves a combination of WT1 and BCG-CWS, or a therapeutic and/orprophylactic agent for cancer which involves a combination of a cancerantigen peptide derived from said WT1 would exert clinical effects as anexcellent anti-tumor-specific immunotherapeutic agent. When WT1 is usedin combination with BCG-CWS, any one of WT1 and BCG-CWS may beadministered first, or they may be mixed together before theadministration.

In this context, cancer antigen peptides derived from WT1 may be thosepeptides found in the amino acid sequence of human WT1 set forth in SEQID NO: 1 which have a motif structure as described above that is boundand presented by an HLA antigen, as well as altered peptides thereof.Particular examples are the peptides listed in Table II to Table XLVI ofWO 2000/18795 and altered peptides based on the motifs, and morepreferable, Arg Met Phe Pro Asn Ala Pro Tyr Leu (SEQ ID NO: 2) and CysMet Thr Trp Asn Gln Met Asn Leu (SEQ ID NO: 3) which have a bindingmotif for HLA-A2 and HLA-A24, or altered peptides thereof based on theHLA-A24 or HLA-A2 binding motif. A specific example of such alteredpeptides is Cys Tyr Thr Trp Asn Gln Met Asn Leu (SEQ ID NO: 4) in whichMet at position 2 of the peptide set forth in SEQ ID NO: 3 has beenreplace by Tyr, an amino acid consistent with the motif.

Regarding the therapeutic and/or prophylactic agent for cancer involvinga combination of WT1 and the bacterium-derived component as describedabove, the administration methods, the doses, the dosage forms and thelike are similar to those described above for the method of inducingantigen-specific T cells.

In connection with these embodiments, the present invention relates to:

a method for enhancing, in a patient, an activity of WT1 protein setforth in SEQ ID NO: 1 or a cancer antigen peptide derived from said WT1protein to induce antigen-specific T cells, which comprisesadministering to said patient a BCG-CWS in an amount effective toenhance the activity to induce antigen-specific T cells;

a method for enhancing, in a patient, an anticancer activity based onthe immunopotentiating action of a BCG-CWS, which comprisesadministering to said patient WT1 protein set forth in SEQ ID NO: 1 or acancer antigen peptide derived from said WT1 protein in an amounteffective to enhance the anticancer activity, wherein the anticanceractivity is enhanced by the activity of the protein or the cancerantigen peptide to induce antigen-specific T cells; preferably themethod wherein the cancer antigen peptide is selected from the groupconsisting of Arg Met Phe Pro Asn Ala Pro Tyr Leu (SEQ ID NO: 2), CysMet Thr Trp Asn Gln Met Asn Leu (SEQ ID NO: 3), and Cys Tyr Thr Trp AsnGln Met Asn Leu (SEQ ID NO: 4); and the method as described above fortreatment and/or prevention of a cancer.

In connection with those embodiments, the invention relates to a use ofa BCG-CWS for preparing a medicament which enhances an activity of WT1protein set forth in SEQ ID NO: 1 or a cancer antigen peptide derivedfrom said WT1 protein to induce antigen-specific T cells; and a use ofWT1 protein set forth in SEQ ID NO: 1 or a cancer antigen peptidederived from said WT1 protein for preparing a medicament which enhancean anticancer activity based on the immunopotentiating action of aBCG-CWS, wherein the anticancer activity is enhanced by the activity ofthe protein or the cancer antigen peptide to induce antigen-specific Tcells; preferably the use wherein the cancer antigen peptide is selectedfrom the group consisting of Arg Met Phe Pro Asn Ala Pro Tyr Leu (SEQ IDNO: 2), Cys Met Thr Trp Asn Gln Met Asn Leu (SEQ ID NO: 3), and Cys TyrThr Trp Asn Gln Met Asn Leu (SEQ ID NO: 4); and the use as describedabove for preparing a medicament for treatment and/or prevention of acancer.

EXAMPLES

The present invention is illustrated below by reference to the followingExamples. The present invention is not, however, limited to suchExamples.

Example 1 Anti-Tumor Effects of a Combination of a WT1 Peptide andBCG-CWS

-   1. Materials and Methods-   1) Cells

C1498, a leukemia cell line, which did not express WT1 derived from aC57BL/6 mouse was purchased from ATCC (Rockville, Md.). C1498(WT1-C1498) cells which expressed mouse WT1 were prepared bytransfecting the C1498 cells with the cDNA of mouse WT1 (WO 00/06602) inthe usual manner.

-   2) Peptide

A 9-mer peptide which represents a cancer antigen peptide derived fromWT1 (sequence: Arg Met Phe Pro Asn Ala Pro Tyr Leu; SEQ ID NO: 2) wassynthesized using Fmoc chemistry on an ABI 430A peptide synthesizer(Applied Biosystems Inc., Foster), and then purified by reverse phasechromatography using a C18 Microbondasphere (Waters Japan, Osaka)column. The synthesized peptide was confirmed on an API IIIE triplequadrupole mass spectrometer (Sciex, Toronto, Canada), and theconcentration was determined by MicroBCA assay (Pierce, Rockford, Ill.)using BSA as a standard.

-   3) Preparation of BCG-CWS

BCG-CWS was used as a non-specific immunopotentiator. BCG-CWS wasformulated into a form of oil-in-water emulsion according to thepreviously report (Cancer Res 24: 121-141, 1979). In brief, 9.6 μl ofsqualane was added to 2 mg of BCG-CWS, and the mixture was homogenized(1200 rpm×1 minute). To the homogenate was added 1 ml of 0.2% Tween80PBS, and the mixture was again homogenized (3000 rpm×8 minutes) to givean emulsion. Recovery rate of BCG-CWS in the emulsion was determined onthe basis of concentration of the galactose that was its sugar chain byphenol-sulfuric acid method, and the emulsion having a recovery rate of30% or more was collected. In the present experiment, the emulsionshaving a recovery rate between 30% and 40% were used.

-   4) Mice

Male C57BL/6 mice, 6 to 8 weeks old (H-2D^(b), H-2K^(b)), were purchasedfrom CLEA Japan, Inc.

-   5) Schedule for Tumor Cell Transplantation and Vaccination

Into mice, 5×10⁵ WT1-C1498 cells were injected intraperitoneally, andvaccination was started on the next day. Into flanks of the mice, 100 μgof BCG-CWS was injected intradermally, and after 24 hours, the WT1peptide was injected intradermally at the same site. Taking theseadministrations as one course, four courses were conducted in total atintervals of one week (FIG. 1).

-   6) Colony Assay

Colony assay was conduced in the usual manner. In brief, myeloid cellsfrom the mice were seeded on α-MEM containing 20% fetal calf serum, 1%bovine serum albumin Fr5, 1.5% methyl cellulose, and various cytokines(100 ng/ml SCF, 10 ng/ml G-CSF) at 3×10⁴ per dish (35 mm), and incubatedfor 14 days, after which colonies having 50 cells or more were counted.

-   7) Detection of WT1-Specific CTL Activity

The spleens were removed from the three mice in which thecoadministration of WT1 peptide and BCG-CWS prohibited any tumor fromestablishing, and the splenocytes were prepared. Similarly, thesplenocytes were also prepared from a control, the untreated mice thathad not been vaccinated. WT1-C1498 cells and C1498 cells were labeledwith ⁵¹Cr. CTL activity was determined using the splenocytes as effectorcells and using the labeled cells as target cells by the ⁵¹Cr releaseassay (J. Immunol., 159: 4753, 1997).

-   2. Results

According to the schedule shown in FIG. 1, tumor cell transplantationand vaccine administration were conducted, and the subsequent formationof tumor masses and the survival rate were observed. FIG. 2 shows thetumor diameters in the mice that were transplanted with 5×10⁵ cells peranimal as observed during the period from the transplantation to Day 65thereafter. Disappearance of the value curve before Day 65 means thatthe animals were dead before Day 65. In the group without anyvaccination, the tumor masses were palpable at the early stage, andrapidly developed. Similar findings were observed in the group receivingthe peptide alone. In the group receiving BCG-CWS alone, the formationof tumor masses was observed to be suppressed, and the rate of tumormass formation was observed slower compared to those observed in thegroups as mentioned above. In the group received the WT1 peptide andBCG-CWS, the formation of tumor masses was further suppressed, and 3 ofthe 4 mice exhibited no palpable tumor masses even 65 days after thetumor cell transplantation.

FIG. 3 shows the curve representing survival rate up to Day 65 from thetumor cell transplantation. All of the mice receiving no vaccinationwere dead on Day 55, and all of the mice receiving the peptide alonewere also dead on Day 58. Contrary to these groups, the mice receivingBCG-CWS alone, and the mice receiving the WT1 peptide and BCG-CWSexhibited survival rates of 60% and 75%, respectively.

FIG. 4 shows the curve representing disease free survival rate up to Day65 from the tumor cell transplantation. The group of the mice receivingthe peptide alone exhibited a disease free survival rate of 0% on Day33, whereas the group of the mice receiving the WT1 peptide and BCG-CWSexhibited a disease free survival rates of 75% even on Day 65, which wasmuch higher.

FIG. 5 shows the results of colony assay of the myeloid cells removed onDay 65 from the mice transplanted with tumor cells and vaccinatedaccording to the schedule of FIG. 1. It was shown that the micevaccinated with WT1 peptide+BCG-CWS and the mice vaccinated with BCG-CWSalone were not significantly different from the mice with neithertransplanted tumor cells nor vaccination in terms of the colony numbersof CFU-GEMM, CFU-GM, CFU-G, CFU-M, and CFU-E. These results show thatCTLs that attack the tumor cells expressing highly WT1 did not affectthe normal cells expressing WT1 (hematopoietic cells in this case).

FIG. 6 shows the results of the experiment examining whether or notWT1-specifc CTLs were induced. The splenocytes from all of the 3 micethat were coadministered with the WT1 peptide and BCG-CWS and that anytumor was not established, killed the WT1-C1498 cells, but exhibited nocytotoxic activity on C1498 cells that did not express WT1. Thesplenocytes from the control, all of the 3 mice without any vaccinationexhibited no cytotoxic activity on WT1-C1498 cells. These show thatadministration of WT1 in combination with BCG-CWS surely induceWT1-specific CTLs.

Industrial Applicability

The method of the present invention enables efficient induction ofantigen-specific T cells. The method of inducing antigen-specific Tcells and related pharmaceutical compositions according to the presentinvention can efficiently, simply and conveniently induceantigen-specific T cells, and are thus useful as anticancer or antiviralagents.

1: A method for inducing antigen-specific T cells in a patientcomprising administering to said patient in need thereof composition (a)which comprises a therapeutically effective amount of an antigen proteinor an antigen peptide as an active ingredient, and composition (b) whichcomprises a therapeutically effective amount of a cell wall skeletonintegrant of the BCG strain of Mycobacterium bovis as an activeingredient, wherein composition (b) is administered in advance and thencomposition (a) is administered. 2: A method according to claim 1,wherein composition (a) is administered about 24 hours after theadministration of composition (b). 3: A method according to claim 1 or2, wherein compositions (a) and (b) are both administered intradermally.4: A method according to claim 3, wherein compositions (a) and (b) areboth administered intradermally at the same site. 5: A method accordingto claim 1, wherein the administration cycle involving compositions (a)and (b) is repeated two or more times. 6: A method according to claim 1,wherein composition (a) comprises a cancer antigen protein or a cancerantigen peptide as an active ingredient. 7: A method according to claim6, wherein the cancer antigen protein or the cancer antigen peptide isWT1 protein set forth in SEQ ID NO: 1 or a cancer antigen peptidederived from said WT1 protein. 8: A method according to claim 7, whereinthe cancer antigen peptide derived from WT1 protein is selected from thegroup consisting of Arg Met Phe Pro Asn Ala Pro Tyr Leu (SEQ ID NO: 2),Cys Met Thr Trp Asn Gln Met Asn Leu (SEQ ID NO: 3), and Cys Tyr Thr TrpAsn Gln Met Asn Leu (SEQ ID NO: 4). 9: A method of treatment and/orprevention of a cancer in a patient which comprises a method accordingto claim
 1. 10: A pharmaceutical composition for enhancing an activityof an antigen protein or an antigen peptide to induce antigen-specific Tcells, which comprises a cell wall skeleton integrant of the BCG strainof Mycobacterium bovis as an active ingredient, and which isadministered before the administration of the antigen protein or theantigen peptide. 11: A pharmaceutical composition for enhancing ananticancer activity based on the immunopotentiating action of a cellwall skeleton integrant of the BCG strain of Mycobacterium bovis, whichcomprises an antigen protein or an antigen peptide as an activeingredient, of which the activity to induce antigen-specific T cellsfacilitates the enhancement of the anticancer activity, said compositionbeing administered after the administration of the cell wall skeletonintegrant of the BCG strain of Mycobacterium bovis. 12: A pharmaceuticalcomposition according to claim 10 or 11, wherein the antigen protein orthe antigen peptide is a cancer antigen protein or a cancer antigenpeptide. 13: A pharmaceutical composition according to claim 12, whereinthe cancer antigen protein or the cancer antigen peptide is WT1 proteinset forth in SEQ ID NO: 1 or a cancer antigen peptide derived from saidWT1 protein. 14: A pharmaceutical composition according to claim 13,wherein the cancer antigen peptide derived from WT1 is selected from thegroup consisting of Arg Met Phe Pro Asn Ala Pro Tyr Leu (SEQ ID NO: 2),Cys Met Thr Trp Asn Gln Met Asn Leu (SEQ ID NO: 3), and Cys Tyr Thr TrpAsn Gln Met Asn Leu (SEQ ID NO: 4). 15: A pharmaceutical compositionaccording to claim 13 for treatment and/or prevention of a cancer. 16:and 17: (Cancelled) 18: A pharmaceutical composition for enhancing anactivity of WT1 protein set forth in SEQ ID NO: 1 or a cancer antigenpeptide derived from said WT1 protein to induce antigen-specific Tcells, which comprises a cell wall skeleton integrant of the BCG strainof Mycobacterium bovis as an active ingredient. 19: A pharmaceuticalcomposition for enhancing an anticancer activity based on theimmunopotentiating action of a cell wall skeleton integrant of the BCGstrain of Mycobacterium bovis, which comprises as an active ingredientWT1 protein set forth in SEQ ID NO: 1 or a cancer antigen peptidederived from said WT1 protein, of which the activity to induceantigen-specific T cells facilitates the enhancement of the anticanceractivity. 20: A pharmaceutical composition according to claim 18 or 19,wherein the cancer antigen peptide derived from WT1 is selected from thegroup consisting of Arg Met Phe Pro Asn Ala Pro Tyr Leu (SEQ ID NO: 2),Cys Met Thr Trp Asn Gln Met Asn Leu (SEQ ID NO: 3), and Cys Tyr Thr TrpAsn Gln Met Asn Leu (SEQ ID NO: 4). 21: (Cancelled) 22: A method forenhancing, in a patient, an activity of WT1 protein set forth in SEQ IDNO: 1 or a cancer antigen peptide derived from said WT1 protein toinduce antigen-specific T cells, which comprises administering to thepatient a cell wall skeleton integrant of the BCG strain ofMycobacterium bovis in an amount effective to enhance the activity toinduce the antigen-specific T cells. 23: A method for enhancing, in apatient, an anticancer activity based on the immunopotentiating actionof a cell wall skeleton integrant of the BCG strain of Mycobacteriumbovis, which comprises administering to the patient WT1 protein setforth in SEQ ID NO: 1 or a cancer antigen peptide derived from said WT1protein in an amount effective to enhance the anticancer activity,wherein the anticancer activity is enhanced by the activity of thecancer antigen protein or the cancer antigen peptide to induceantigen-specific T cells. 24: A method according to claim 22 or 23,wherein the cancer antigen peptide derived from WT1 protein is selectedfrom the group consisting of Arg Met Phe Pro Asn Ala Pro Tyr Leu (SEQ IDNO: 2), Cys Met Thr Trp Asn Gln Met Asn Leu (SEQ ID NO: 3), and Cys TyrThr Trp Asn Gln Met Asn Leu (SEQ ID NO: 4). 25: A method according toclaim 22 for treatment and/or prevention of a cancer. 26: through 29:(Cancelled)